Proteins synthesized in the cytoplasm must be sorted to other parts of the eukaryotic cell to function. Most have a single destination but there is a unique subset of proteins which must be partitioned between two cellular compartments. The TRM1 and MOD5 gene products are tRNA modification enzymes that are dually localized in the yeast Saccharomyces cerevisiae. The TRM1 gene product is the only known example of a gene that codes for proteins destined to the mitochondria and nucleus while the MOD5 product is mitochondrial and cytoplasmic, not nuclear. The objective of the proposed studies is to understand the mechanisms that result in the dual localization of these two gene products. First, we will determine how the enzymes found in the different compartments are synthesized from the genes encoding them. Second, molecular biological and cell biological methods will be used to determine what portions of the proteins contain signals necessary to target them to the mitochondria and the nucleus and whether the absence of these signals leads to cytoplasmic accumulation. Third, we will determine how a protein with both mitochondrial and nuclear targeting signals can be localized exclusively to mitochondria. Fourth, we will use genetic approaches to address the questions of why proteins are sequestered to certain organelles. Finally, we will attempt to identify genes encoding soluble factors and membrane receptors that comprise the cellular machinery essential for proper localization of MOD5 and TRM1 proteins.